A medium for culturing microorganisms can be prepared by dispersing gelling agent in an aqueous solution containing nutrients and other ingredients necessary for the growth of microorganisms. Unfortunately, the use of conventional gelling agents is often inconvenient for the end-user. For example, when carrying out standard "plate count" or "pour plate" methods to determine the number of microorganisms in a liquid sample such as water or milk, the use of agar as a gelling agent is particularly inconvenient and time-consuming. The agar-containing medium, which has generally been prepared in bulk and sterilized ahead of time, must be melted, for example, in boiling water or by exposure to steam. The hot medium must then be carefully cooled to approximately 45.degree. C. A series of dilutions of a test sample is then prepared and an aliquot of each dilution is placed in a petri dish. The partially cooled, but still liquefied, medium is then poured into each dish, mixed with the aliquot of test sample, and allowed to solidify. After incubation, the colonies growing in each dish are counted by visual inspection. In this manner the number of colony-forming units ("CFU", i.e., microorganisms capable of forming colonies) originally present in the test sample can be determined.
Simpler methods exist, wherein the need for the end-user to manipulate the agar-containing medium can be eliminated, and significantly less medium need be used. For example, U.S. Pat. No. 4,565,783 discloses a device that provides the user the opportunity to perform standard plate counts in bacterial assays without manipulating an agar-containing medium. This device contains a powdered dry culture medium adhered to a waterproof substrate. Upon inoculation of the device with an aqueous sample suspected of containing microorganisms, the medium is reconstituted to form a homogeneous gel-like medium suitable for quantitative culturing of the microorganisms present in the sample. One such device (Petrifilm.TM. brand growth medium, available from 3M Company, St. Paul, Minn.) is particularly useful for the assay of bacteria.
In various industries, such as the dairy, fruit juice, wine, and beer industries, there exists a need to assay not only bacteria but also molds. The art does not typically distinguish mold assays from bacterial assays when devices for the assay of microorganisms are discussed, but it is known to those skilled in the art that mold assays are often more difficult than bacterial assays. One reason for this difficulty is that some molds require more oxygen for growth than do most bacteria, and thus some devices (e.g., devices that are covered or otherwise sealed off from outside air, as are many devices useful for bacterial assays) are unsuitable for use with molds. Also, some molds tend to grow homogeneously throughout a culture medium rather than in discrete, countable colonies.
Devices and methods for the growth of microorganisms known in the art generally involve incubation either in an environment, open to air (e.g., in a petri dish), in order to allow an adequate supply of air to the medium, or in a sealed environment to prevent desiccation of the medium. Either approach is generally satisfactory when the devices use relatively large amounts of water and medium. Where relatively small amounts of water and medium are used, however, such as with Petrifilm.TM. brand growth medium, the user is faced with the following problem: if the device is allowed an adequate supply of air, the water can evaporate, thereby drying out the medium during incubation; on the other hand, if the device is sealed off, the air supply might be insufficient to sustain growth of microorganisms that require more oxygen.
One approach to the problem is seen in the Millipore Corp. (Bedford, Mass.) "Yeast and Mold Swab Sampler" used for the analysis of aqueous solutions. This device consists of a plastic tab supporting a microporous filter bonded to an absorbent pad that contains dehydrated nutrient medium. An aqueous sample passes through the filter into the pad, thereby isolating any microorganisms on the filter while hydrating the absorbent pad and the nutrient medium. The device is then placed in a container, with the plastic tab serving to seal the container. There is sufficient air in the container to support the growth of yeasts and molds. Nutrients are intended to pass from the medium into the filter, thus allowing the growth of microorganisms in the filter.
Certain other devices and methods that involve a water- or nutrient-permeable barrier (e.g., a membrane) to microorganisms are known to be useful for the growth of microorganisms. For example, U.S. Pat. Nos. 3,814,670, 3,843,452, and 4,250,256, disclose such devices. In no such device, however, does it appear that the barrier itself is used to provide an adequate supply of air to the growing microorganisms.